Enzyme and Prebiotic Combinations for Enhancing Probiotic Efficacy

ABSTRACT

This disclosure relates to enhancing growth and/or activity of lactobacilli using a prebiotic formulation which includes iso-malto oligosaccharides and α-galactosidase; and to enhancing growth and/or activity of bifidobacteria using a prebiotic formulation which includes iso-malto oligosaccharides and β-glucanase. Other combinations of fibers and enzymes are described below which also stimulate growth and activity of lactobacilli or bifidobacteria. These combinations of enzymes and prebiotics can be taken separately or added to foods, including desserts.

RELATED APPLICATIONS

This application claims priority to and is a continuation-in-part ofU.S. application Ser. No. 13/267,042, filed Oct. 6, 2011, which is acontinuation of U.S. application Ser. No. 12/022,380 (U.S. Pat. No.8,066,986), filed Jan. 30, 2008, which is a nonprovisional of U.S.Provisional No. 60/887,628, filed Feb. 1, 2007, and is also acontinuation-in-part of U.S. application Ser. No. 13/118,550, filed May30, 2011 which is a nonprovisional of U.S. Provisional No. 61/363,172,filed Jul. 9, 2010.

FIELD OF THE APPLICATION

The field is enzymes and prebiotics for enhancing probiotic efficacy.

BACKGROUND

Lactic acid producing bacteria that are capable of improving ormaintaining intestinal health and function, including reducingconstipation (primarily from the Lactobacillus and Bifidobacteriumgenera) are termed probiotic bacteria. Dietary supplements withprobiotic bacteria as the active ingredient currently enjoy sales ofover $700 million annually, and the market growth is approaching 30%annually. The other piece of the probiotic market is probiotic foods,especially yogurt and desserts. This segment of the market is over $1Billion annually.

The reported health benefits of probiotics include supporting the immunesystem (inhibiting allergic response and neoplastic growth), treatinginflammatory bowel disease, offsetting lactose intolerance, and reducingcholesterol. They are also useful for repopulating the gut afterantibiotic therapy. Probiotic growth in the intestinal tract, followingingestion, depends to a large extent on the nutrients present in thepatient's diet. Typical human diets are not well suited for probioticsand, given the abundance of and competition from many less fastidiousdigestive tract bacteria (including pathogenic strains such asclostridium, rotaviruses, pathogenic E. coli and Helicobacter pylori) itcan be difficult for probiotics to effectively multiply in vivo. To helpcorrect this problem, manufacturers of probiotic dietary supplementsoften include prebiotics (nutrient substances that encourage the growthof probiotics in vivo) in their formulations.

Many types of prebiotics are not digested or absorbed in the smallintestine but pass into the colon where they stimulate the growth ofprobiotic bacteria. Fructo-oligosaccharides (FOS) are one type ofprebiotic; inulin compounds (which are also oligosaccharides) areanother. For these compounds to be effective they must be ingested inrelatively large quantities, such as 4-10 grams/day for FOS and 10-14grams/day for inulin. Probiotics, by comparison, can be effectivelyadministered in milligram quantities, containing 10⁷-10¹⁰ colony formingunits (cfu). Thus, it is impractical to mix FOS or inulin withprobiotics and deliver them in capsules or tablets. Further, suchcarbohydrate type prebiotics often break down to glucose, in vivo, whichenhances growth of non-probiotic bacteria, including pathogenicclostridium. Moreover, FOS can cause flatulence and abdominal pain andsome people experience severe allergic reactions to inulin. Therefore,there is a need for a non-carbohydrate prebiotic that can be used at lowdosage while effectively stimulating probiotic bacteria.

Although enzymes have been used to generate prebiotics under laboratoryconditions followed by subsequent feeding of the preformed prebiotics toachieve probiotic stimulation (see U.S. Pat. Nos. 6,791,015 and6,730,502), no one has suggested using enzymes to generate these effectsin vivo. U.S. Pat. No. 5,817,350 discloses the use of the prebioticenzymes cellulase, amylase and hemicellulase, for use as dietarysupplements, but not use of these enzymes to stimulate administeredprobiotics. Enzymes which can generate compounds which significantlyincrease probiotic growth or activity without generating significantamounts of glucose or otherwise stimulating growth of undesirabledigestive tract bacteria, would be a significant improvement overexisting formulations.

Iso-malto oligosaccharides can be enzymatically digested to simplersugars by inulinase, which is included in some commercially-availableprobiotic formulations because it digests linear fructans (inulin).Inulin is known to stimulate bifidobacteria growth. Inulin in diet doesnot lead to a rise in serum glucose or stimulate insulin secretion, butinulinase digestion generates significant fructose. It is not clearwhether fructose would preferentially increase growth of probiotics orof competitive digestive tract bacteria, including pathogenic bacteria.

The product Beano™ includes the enzyme alpha-galactosidase, which canbreak down polysaccharides and oligosaccharides, including iso-maltooligosaccharides, which are in foods such as legumes (beans and peanuts)and cruciferous vegetables (cauliflower, broccoli, cabbage, brusselssprouts, among others). The enzyme breaks those complex sugars intosimpler sugars, making these foods somewhat more digestible, and therebyreducing intestinal gas. Beano does not include any probiotics in itsformulation.

The hydrolysis of lactose to glucose and galactose is catalyzed by theenzymes lactase and β-galactosidase. Because β-galactosidase wouldgenerate glucose from lactose in the diet, it is not preferred forinclusion in probiotics. Lactobacillus bulgaricus producesbeta-galactosidase, and this strain is a probiotic purported to treatlactose intolerance.

SUMMARY

This disclosure relates to enhancing growth and/or activity oflactobacilli using a prebiotic formulation which includes iso-maltooligosaccharides and α-galactosidase; and to enhancing growth and/oractivity of bifidobacteria using a prebiotic formulation which includesiso-malto oligosaccharides and β-glucanase. Other combinations of fibersand enzymes are described below which also stimulate growth and activityof lactobacilli or bifidobacteria.

The enzymes α-galactosidase and β-glucanase react with the fiberprebiotics to generate shorter chain oligosaccharides, some of which arepreferential growth enhancers for the probiotics. The enzymes arebelieved to not generate significant amounts of glucose in the reaction,as it can stimulate growth of undesirable bacterial species.

The fiber prebiotic, the enzyme(s) and the probiotic(s) can beadministered in a combined formula, or, the fiber prebiotic with theappropriate enzyme (e.g., α-galactosidase or β-glucanase) can be inmedia where they can react (e.g., added to foods) and the probiotic canbe administered separately. Or, each of these ingredients can beadministered separately, whereby the prebiotic and the enzyme can reactin vivo, and the probiotic can metabolize the reaction product(s) toenhance its growth and activity.

More specifically, the invention relates to enhancing in vivo growthand/or activity of both lactobacilli and bifidobacteria using iso-maltooligosaccharides as the prebiotic, and both α-galactosidase andβ-glucanase as the enzymes. Again, these ingredients can be combined oradministered separately.

Pectinase may also be included in any of the formulations describedherein. Pectinase are a class of enzymes including pectolyase, pectozymeand polygalacturonase. They break down pectin, a polysaccharide found inthe cell walls of plants.

The above formulas could also include other prebiotics (includinginulin, wheat dextrin, and partially hydrolyzed guar gum (“PHGG”)) andother fiber-digesting enzymes, including Fiberase™ (a combination ofcellulase, hemicellulase, pectinase and xylanase). Cellulase includescellulase-TL and cellulase-AN. The formulas could also include proteaseenzymes including papain, bromelain, fungal protease, fungalacid-protease, bacterial protease, fungal peptidase, nattokinase,serapeptase, trypsin, chymotrypsin pancreatin and pepsin. Carbohydraseenzymes (including alpha-amylase, amylase, glucoamylase, lactase, andinvertase) are generally not preferred in the formula, as they generateglucose.

The above formulas could also include sunflower lecithin and/or oleicacid (as described in U.S. Pat. No. 8,105,577, incorporated byreference) and/or the food grade polysorbate surfactants (as describedin U.S. Pat. No. 8,066,986, incorporated by reference): Polysorbate-60,polysorbate-80 or any polysorbate with an HLB>12, where HLB is thehydrophile-lipophile balance, designated from 1 to 20.

The above formulas could also contain other carriers, binders oradsorbents, including but not limited to food grade starches andsilicates. The above formulas can be packaged for administration incapsules, tablets or packets, or combinations thereof. Alternatively,they can be added to foods, separately or in combination.

Additional combinations of substrates, enzymes and probiotics whichenhanced growth and/or activity of the probiotics are described belowand are also within the scope of the inventions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the growth of BL-04 (Bifidobacterium lactis) in MRS broth(“Control (scratch)”) made by the experimenter to have the sameconstituents as commercial MRS broth (by Difco™), and its growth inmodified MRS broth where the glucose has been removed and a quantity offiber (in this case, VitaFiber™, by BioNeutra) has been added which isapproximately the same as the amount of glucose removed (hereinafter,such modified MRS broth is referred to as “No-G-Broth”).

FIG. 1B shows the activity of BL-04 (Bifidobacterium lactis) in MRSbroth, and in No-G-Broth with added VitaFiber™.

FIG. 2A shows the growth of NCFM (Lactobacillus acidophilus) in MRSbroth, and in No-G-Broth with added VitaFiber™ as in FIGS. 1A, 1B.

FIG. 2B shows the activity of NCFM (Lactobacillus acidophilus) in MRSbroth, and in No-G-Broth with added VitaFiber™ as in FIGS. 1A, 1B.

FIG. 3A shows the growth of BL-04 (Bifidobacterium lactis) in No-G-Brothwith added VitaFiber™ and where VitaFiber™ was pre-digested withcellulase before the remaining No-G-Broth broth ingredients were added.

FIG. 3B shows the activity of BL-04 (Bifidobacterium lactis) inNo-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digestedwith cellulase before the remaining No-G-Broth broth ingredients wereadded.

FIG. 4A shows the growth of NCFM (Lactobacillus acidophilus) inNo-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digestedwith pectinase before the remaining No-G-Broth broth ingredients wereadded.

FIG. 4B shows the activity of NCFM (Lactobacillus acidophilus) inNo-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digestedwith pectinase before the remaining No-G-Broth broth ingredients wereadded.

FIG. 5A shows the growth of BL-04 (Bifidobacterium lactis) in No-G-Brothwith added VitaFiber™, and where VitaFiber™ was pre-digested with eitherβ-glucanase or α-galactosidase before the remaining No-G-Broth brothingredients were added. The Vitafiber™ was sterilized by filtering itthrough a sterile 0.22 μm filter, while the MRS broth ingredients in theVitaFiber™ mixture were separately sterilized by autoclaving.

FIG. 5B shows the activity of BL-04 (Bifidobacterium lactis) in the sameconditions and media as in FIG. 5A.

FIG. 6A shows the growth of NCFM (Lactobacillus acidophilus) inNo-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digestedwith either β-glucanase or α-galactosidase before the remainingNo-G-Broth broth ingredients were added.

FIG. 6B shows the activity of NCFM (Lactobacillus acidophilus) in thesame conditions and media as in FIG. 6A.

FIG. 7A shows the growth of BL-04 (Bifidobacterium lactis) in No-G-Brothwhere a quantity of partially hydrolyzed guar gum (“PHGG”) has beenadded which is approximately the same as the amount of glucose removedfrom the starting MRS broth; and where PHGG was pre-digested with eitherβ-glucanase or α-galactosidase before the remaining No-G-Broth brothingredients were added.

FIG. 7B shows the activity of BL-04 (Bifidobacterium lactis) in the sameconditions and media as in FIG. 7A.

FIG. 8A shows the growth of NCFM (Lactobacillus acidophilus) inNo-G-Broth where a quantity of partially hydrolyzed guar gum (“PHGG”)has been added which is approximately the same as the amount of glucoseremoved from the starting MRS broth; and where PHGG was pre-digestedwith either β-glucanase or α-galactosidase before the remainingNo-G-Broth broth ingredients were added.

FIG. 8B shows the activity of NCFM (Lactobacillus acidophilus) in thesame conditions and media as in FIG. 8A.

FIG. 9A shows the growth of LP-115 (Lactobacillus plantarum) inNo-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digestedwith either with either pectinase or a 50:50 mixture of α-galatosidaseplus pectinase before the remaining No-G-Broth ingredients were added.Note that “cold filter” means the VitaFiber™ was sterilized by filteringit through a sterile 0.22 μm filter, while the MRS broth ingredients inthe VitaFiber™ mixture were separately sterilized by autoclaving.

FIG. 9B shows the activity of LP-115 (Lactobacillus plantarum) in thesame conditions and media as in FIG. 9A.

FIG. 10A shows the growth of NCFM (Lactobacillus acidophilus) whereVitaFiber™ was pre-digested with α-galactosidase before the remainingNo-G-Broth ingredients (except polysorbate 80) were added along withadded LactoStim™ (sunflower lecithin and oleic acid). VitaFiber™ wasalso pre-digested with α-galactosidase plus pectinase, before theremaining No-G-Broth ingredients were added.

FIG. 10B shows the activity of NCFM (Lactobacillus acidophilus) in thesame conditions and media as in FIG. 10A.

FIG. 11A shows the growth of BL-04 (Bifidobacterium lactis) whereVitaFiber™ was pre-digested with β-glucanase before the remainingNo-G-Broth ingredients (except polysorbate 80) were added along withadded LactoStim™, and where VitaFiber™ was pre-digested with β-glucanaseand cellulase before the remaining No-G-Broth ingredients were added.

FIG. 11B shows the growth of BL-04 (Bifidobacterium lactis) in the sameconditions and media as in FIG. 11A.

FIG. 12A shows the growth of BL-04 (Bifidobacterium lactis) inNo-G-Broth where a quantity of inulin has been added which isapproximately the same as the amount of glucose removed from thestarting MRS broth, and where inulin was pre-digested with β-glucanasebefore the remaining No-G-Broth ingredients were added.

FIG. 12B shows the activity of BL-04 (Bifidobacterium lactis) in thesame conditions and media as in FIG. 12A.

FIG. 13A shows the growth of NCFM (Lactobacillus acidophilus) inNo-G-Broth with inulin substituted for glucose, and where inulin waspre-digested with α-galactosidase before the remaining No-G-Brothingredients (except polysorbate 80) were added along with addedLactoStim™.

FIG. 13B shows the activity of NCFM (Lactobacillus acidophilus) in thesame conditions and media as in FIG. 13A.

FIG. 14A shows the growth of BL-04 (Bifidobacterium lactis) inNo-G-Broth with VitaFiber™ substituted for glucose, and where VitaFiber™was pre-digested with one of two different concentrations of β-glucanasebefore the remaining No-G-Broth ingredients were added.

FIG. 14B shows the activity of BL-04 (Bifidobacterium lactis) in thesame conditions and media as in FIG. 14A.

FIG. 15A shows the growth of NCFM (Lactobacillus acidophilus) inNo-G-Broth with VitaFiber™ substituted for glucose, and where VitaFiber™was pre-digested with one of: (i) α-galactosidase; (ii) a lowerconcentration of α-galactosidase and β-glucanase; and (iii) a higherconcentration of α-galactosidase and β-glucanase.

FIG. 15B shows the activity of NCFM (Lactobacillus acidophilus) in theconditions and same media as in FIG. 15A.

FIG. 16A shows the growth of BL-04 and NCFM (i) in MRS broth, (ii) inNo-G-Broth with VitaFiber™ substituted for glucose, (ii) whereVitaFiber™ was pre-digested with a 1:3 blend of β-glucanase andα-galactosidase before (a) adding the other ingredients in No-G-Broth,(b) adding LactoStim™ and the other ingredients in No-G-Broth but notpolysorbate 80.

FIG. 16B shows the activity of BL-04 and NCFM in the same conditions andmedia as in FIG. 16A.

FIG. 17A shows the growth of Latobacillus salivarius (LS-33) whereVitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii)β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase;before adding LactoStim™ and the other ingredients in No-G-Broth but notpolysorbate 80.

FIG. 17B shows the activity of Latobacillus salivarius (LS-33) in thesame conditions and media as in FIG. 17A.

FIG. 18A shows the growth of Lactobacillus paracasei (LPC-37) whereVitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii)β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase,before adding LactoStim™ and the other ingredients in No-G-Broth but notpolysorbate 80.

FIG. 18B shows the activity of Lactobacillus paracasei (LPC-37) in thesame conditions and media as in FIG. 18A.

FIG. 19A shows the growth of Lactobacillus plantarum (LP-115) whereVitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii)β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidasebefore adding LactoStim™ and the other ingredients in No-G-Broth but notpolysorbate 80.

FIG. 19B shows the activity of Lactobacillus plantarum (LP-115) in thesame conditions and media as in FIG. 19A.

FIG. 20A shows the growth of Lactobacillus rhamnosus (Lr-32) whereVitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii)β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidasebefore adding LactoStim™ and the other ingredients in No-G-Broth but notpolysorbate 80.

FIG. 20B shows the activity of Lactobacillus rhamnosus (Lr-32) in thesame conditions and media as in FIG. 20A.

FIG. 21A shows the growth of Bifidobacterium lactis (Bi-07) whereVitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii)β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase,before adding LactoStim™ and the other ingredients in No-G-Broth but notpolysorbate 80.

FIG. 21B shows the activity of Bifidobacterium lactis (Bi-07) in thesame conditions and media as in FIG. 21A.

FIG. 22A shows growth of Lactobacillus rhamnosus (LR-32) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested withα-galatosidase before adding the other ingredients in No-G-Broth.

FIG. 22B shows the activity of Lactobacillus rhamnosus (LR-32) in thesame conditions and media as in FIG. 22A.

FIG. 23A shows the growth of Lactobacillus salivarius (LS-33) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested withα-galatosidase before adding the other ingredients in No-G-Broth.

FIG. 23B shows the activity of Lactobacillus salivarius (LS-33) in thesame conditions and media as in FIG. 23A.

FIG. 24A shows the growth of Lactobacillus acidophilus (NCFM) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested withα-galatosidase before adding the other ingredients in No-G-Broth.

FIG. 24B shows the activity of Lactobacillus acidophilus (NCFM) in thesame conditions and media as in FIG. 24A.

FIG. 25A shows the growth of Bifidobacterium lactis (BL-04) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested withβ-glucanase before adding the other ingredients in No-G-Broth.

FIG. 25B shows the activity of Bifidobacterium lactis (BL-04) in thesame conditions and media as in FIG. 25A.

FIG. 26A shows the growth of Bifidobacterium lactis (Bi-07) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested withβ-glucanase before adding the other ingredients in No-G-Broth.

FIG. 26B shows the activity of Bifidobacterium lactis (Bi-07) in thesame conditions and media as in FIG. 26A.

FIG. 27A shows the growth of Bifidobacterium breve (BB-03) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested withβ-glucanase before adding the other ingredients in No-G-Broth.

FIG. 27B shows the activity of Bifidobacterium breve (BB-03) in the sameconditions and media as in FIG. 27A.

FIG. 28A shows the growth of Lactobacillus plantarum (LP-115) with WheatDextrin in No-G-Broth, and where Wheat Dextrin was pre-digested witheither pectinase or α-galactosidase before adding the other ingredientsin No-G-Broth.

FIG. 28B shows the activity of Lactobacillus plantarum (LP-115) in thesame conditions and media as in FIG. 28A.

DETAILED DESCRIPTION

Certain enzymes, acting upon certain fiber sources, render the fibersources a preferential food source (prebiotic) for probiotic bacteria.As shown in the figures, different species of probiotics, all of whichare lactic acid producing bacteria, respond differently to variousenzymes and fiber sources. All of the enzymes described are“fiber-digesting” enzymes, which render complex oligosaccharides intosimpler oligosaccharides, but without significant production of glucose.Particular enzyme/fiber combinations respectively improve both activityand growth of Lactobacillus and of Bifidobacteria.

These combinations of enzymes and prebiotics can be used to improve thecommercial value and performance of probiotic products, which allinclude Lactobacillus and/or Bifidobacteria. These combinations ofenzymes and prebiotics can be formulated with Lactobacillus and/orBifidobacteria, e.g., in a capsule or tablet form. Another use for themwould be as food additives to foods that do not requiringheating/boiling before consumption, e.g., yogurt, ice cream, desserts,bread or other bakery goods, snacks, breakfast cereal or candy.

These combinations of enzymes and prebiotics could be added to suchfoods with or without probiotics, and with or without other growthstimulants for probiotics (e.g., polysorbate 80, sunflower lecithin oroleic acid). If such combinations of enzymes and prebiotics were added,for example, to yogurt, they could act to produce the less complexoligosaccharides after consumption. If the probiotics (with or withoutother growth stimulants) are ingested near the time the yogurt isconsumed, they could metabolize the less complex oligosaccharidespresent, and thereby have their growth and activity stimulated.Alternatively, the probiotics (with or without other growth stimulants)can be directly added to such foods along with the appropriatecombination of enzymes and prebiotics, where they can stimulateprobiotic growth after consumption.

The combinations of enzymes and fiber sources which were shown tosignificantly enhance growth and activity of particular probioticswithout glucose in the growth media are (note that VitaFiber™ issubstantially isomalto-oligosaccharide, as shown in Table 1):

α-galactosidase with isomalto-oligosaccharide enhanced growth andactivity of lactobacillus (see FIGS. 6A; 6B; FIGS. 9A; 9B; 10A; 10B;15A; 15B; 16A; 16B; 17A, 17B-20A, 20B);

β-glucanase with isomalto-oligosaccharide enhanced growth and activityof bifidobacterium (see FIGS. 5A, 5B; 11A; 11B; 16A; 16B; 14A; 14B and21A, 21B);

α-galactosidase with partially hydrolyzed guar gum enhanced growth andactivity of bifidobacterium (see FIGS. 7A; 7B);

β-glucanase with partially hydrolyzed guar gum enhanced growth andactivity of lactobacillus (see FIGS. 8A, 8B);

α-galactosidase with partially hydrolyzed guar gum enhanced growth andactivity of lactobacillus (see FIGS. 8A, 8B);

α-galactosidase with inulin enhanced growth and activity oflactobacillus (see FIGS. 13A, 13B);

α-galactosidase with wheat dextrin enhanced growth and activity oflactobacillus (see FIGS. 22A, 22B-24A, 24B; 28A, 28B);

pectinase with wheat dextrin enhanced growth and activity oflactobacillus (see FIGS. 28A, 28B);

pectinase with isomalto-oligosaccharide enhanced growth and activity oflactobacillus (see FIGS. 4A; 4B; 9A; 9B; 10A; 10B);

β-glucanase with wheat dextrin enhanced growth and activity ofbifidobacterium (see FIGS. 25A; 25B-27A; 27B);

cellulase with isomalto-oligosaccharide enhanced growth and activity ofbifidobacterium (see FIGS. 3A; 3B)

Tables 1, 2 and 3 below specify the fiber sources, probioticspecies/strains, and the enzymes used in the Examples, which generatedthe results shown in the figures.

TABLE 1 Fiber Brand Source Partially Hydrolyzed Sunfiber ® TaiyoInternational Guar Gum (PHGG) Isomalto-oligosaccharide VitaFiber ™BioNeutra Inulin Oliggo-Fiber ™ Instant Cargill Wheat DextrinBenefiber ® Novartis

TABLE 2 Probiotic Strain Designation Source Bifidobacterium lactis BL-04(BL-34) Danisco/DuPont Bifidobacterium lactis Bi-07 Danisco/DuPontLactobacillus acidophilus NCFM (LA-1) Danisco/DuPont Lactobacillusparacasei LPC-37 (F-19) Danisco/DuPont Lactobacillus rhamnosus Lr-32(LR-44) Danisco/DuPont Lactobacillus plantarum LP-115 (LP-29)Danisco/DuPont Lactobacillus salivarius LS-33 (LS-30) Danisco/DuPontBifidobacterium breve BB-03 Danisco/DuPont

TABLE 3 Enzyme Units Source Cellulase 150,000 CU/gm BioCat Hemicellulase400,000 HCU/gm BioCat Pectinase 3,500 endo-PG/gm BioCat Xylanase 150,000XU/gm BioCat β-Glucanase 3,000 BGU/gm BioCat α-Galactosidase 15,000GALU/gm BioCat Fiberase (CHPX) BioCat Cellulase 48,000 CU/gmHemicellulase 102,400 HCU/gm Pectinase 420 endo-PG/gm Xylanase 25,000XU/gm

Tables 3A and 3B below explain the units used in Table 3 above.

TABLE 3A Enzyme Unit Abbreviation Enzymatic Unit Reference Method CUCellulase unit FCC 8^(th) Edition HCU Hemicellulase unit FCC 8^(th)Edition endo-PG endo-Polygalacturonase unit Genencor InternationalProcedure No. ME 400.39, 1981 XU Xylanase unit 1989. Appl. Microbiol.Biotechnol.30: 5-10 BGU β-Glucanase unit Novozymes, EB-0338.02/01 GALUα-Galactosidase unit FCC 8^(th) Edition

TABLE 3B Enzymatic Unit Definition Cellulase unit The amount of activitythat will produce a relative fluidity change of 1 in 5 minutes in adefined carboxymethyl cellulose substrate under the conditions of theassay at 40° C. Hemicellulase That activity that will produce a relativefluidity unit change of 1 over a period of 5 minutes in a locust beangum substrate. Endo- The amount of enzyme that reduces the viscosityPolygalacturonase of the pectin solution by 50% per minute under unitthe conditions of the assay. Xylanase unit The quantity of enzyme thatwill liberate 1 μmol per minute of xylose from wheat arabinoxylan underdefined conditions of temperature and pH. β-Glucanase unit The amount ofenzyme which liberates glucose to 1 μmol glucose per minute.α-Galactosidase The quantity of enzyme that will liberate 1 μmol perunit minute of p-nitrophenol under the conditions of the assay.

Preparing the Growth Media

For the growth and activity determinations described below and shown inthe figures, the starting media composition was MRS broth, which wasmodified as described below. MRS broth (including the MRS broth labeled“control-scratch” in FIGS. 1A, 1B, 2A, 2B) consisted of the following:

Proteose Peptone #3 4.0 gms Beef Extract 4.0 gms Yeast Extract 2.0 gmsGlucose (or substitute ingredient) 8.0 gms Polysorbate 80 (orLactoStim ®) 0.4 gms Ammonium Citrate 0.8 gms Sodium Acetate 2.0 gmsMagnesium Sulfate 0.04 gms Manganese Sulfate 0.02 gms DipotassiumPhosphate 0.8 gms DI Water 400 mls

As noted, the starting MRS broth (by Difco™), included glucose. Theglucose was removed to generate No-G-Broth, and then an equivalentquantity of one of the fiber sources in Table 1 (i.e., VitaFiber™, PHGG,Inulin or Wheat Dextrin) was added into the No-G-Broth broth, togenerate each In different formulations noted in the figures and theirdescription. In cases where an enzyme is included in the formulation inthe figures and their description, the enzyme and the fiber were firstreacted, then the remaining ingredients in the No-G-Broth were added (asdescribed further below). In cases where LacoStim™ is included,following the reaction between the enzyme and fiber, the remainingingredients in No-G-Broth were added, but not polysorbate 80.

To determine growth and activity with different media, enzymes andbacterial strains, the fiber source was substituted for glucose in MRSbroth and filled into 500 ml flasks, which were then autoclaved at 121°C. for 15 minutes. Each flask was tempered to 37° C. and asepticallyinoculated with 0.14 gram (Table 4) of one of the freeze-dried probioticstrain(s) listed in Table 2. The CFUs (colony forming units) of 0.14gram of each strain in Table 2 is shown in the right-hand column inTable 4.

TABLE 4 Probiotic species Strain CFUs from 0.14 gram Bifidobacteriumlactis BL-04 (BL-34) 134.4 × 10⁹ Bifidobacterium lactis Bi-07 92.4 × 10⁹Lactobacillus acidophilus NCFM (LA-1) 54.6 × 10⁹ Lactobacillus paracaseiLPC-37 (F-19)   42 × 10⁹ Lactobacillus rhamnosus Lr-32 (LR-44) 31.5 ×10⁹ Lactobacillus plantarum LP-115 (LP-29) 88.9 × 10⁹ Lactobacillussalivarius LS-33 (LS-30) 88.2 × 10⁹ Bifidobacterium breve BB-03   42 ×10⁹

At specific time intervals, a 30 ml sample from each flask wasaseptically transferred into a HACH 2100N Turbidimeter cell. Theturbidity of each sample was read and the turbidity results werereported in NTU's, where greater turbidity indicates greater growth. Thesame 30 ml sample that was used for the turbidity reading wastransferred into a 250 ml glass beaker, and the pH was recorded. Thesample was then titrated using 0.1 N NaOH to an end point of pH 6.8, andthe quantity of NaOH used was recorded. The % Lactic Acid was calculatedusing the following formula:

${\% \mspace{14mu} {Lactic}{\mspace{11mu} \;}{Acid}} = \frac{\left( {\left( {{mls}\mspace{14mu} {of}\mspace{14mu} 0.1N{\mspace{11mu} \;}{NaOH}} \right) \times \left( {90{\mspace{11mu} \;}{{gm}/{mole}}} \right) \times \left( {1\mspace{14mu} {L/1000}\mspace{14mu} {ml}} \right)} \right) \times 100}{{mls}\mspace{14mu} {of}\mspace{14mu} {sample}}$${\% \mspace{14mu} {Lactic}{\mspace{11mu} \;}{Acid}} = \frac{\left( {{mls}\mspace{14mu} {of}\mspace{14mu} 0.1N\mspace{14mu} {NaOH}} \right) \times (0.9)}{30}$

Higher % Lactic acid indicates higher activity. For samples that werepre-digested with enzymes, the fiber source (table 1) was added to 400mls of de-ionized water along with the enzyme(s) and incubated for 24hours in a 37° C. water bath. The remaining MRS components (as specifiedin the figures and their description) were then added to each flask andautoclaved at 121° C. for 15 minutes. Each flask was tempered to 37° C.and aseptically inoculated with 0.14 gram of the specified probioticstrain(s). At specific time intervals, 30 ml samples were asepticallytaken and the turbidity, pH and % Lactic Acid was determined for eachflask as described above. All probiotics were held at −10° F. prior touse. All enzymes were held at 5° C. prior to use.

Example #1

Isomalto-oligosaccharide prebiotic (VitaFiber™) was substituted as thecarbohydrate source in MRS broth, replacing glucose. Growth ofBifidobacterium lactis (BL-04) and Lactobacillus acidophilus (NCFM) weremonitored. BL-04 grew better with the isomalto-oligosaccharide than withglucose (FIG. 1A). NCFM growth was stimulated by theisomalto-oligosaccharide, however, not as much as with the glucosecontrol (FIG. 2A).

Example #2

Isomalto-oligosaccharide (VitaFiber™) was digested for 24 hours in a 37°C. water bath with 0.2% (w/v) of various enzymes. Enzymes tested wereeither Fiberase™ (which is a combination of cellulase, hemicellulase,pectinase and xylanase), and cellulase, hemicellulase, pectinase andxylanase were also tested individually. Digesting VitaFiber™ (VF) withFiberase™, cellulase, hemicellulase or xylanase gave a higher activityfor BL-04 than undigested VF. Pectinase did not. The highest activityoccurred with cellulase (1.368% lactic acid) (FIG. 3B). All flasks withenzyme digested fiber and NCFM had a higher activity than the undigestedfiber control. In the case of NCFM, pectinase (FIG. 4B) gave the highestactivity with 1.350% lactic acid produced.

Example #3

VitaFiber™ (VF) was digested with either 0.2% (wt/vol) β-glucanase or0.2% (wt/vol) α-galactosidase and inoculated with either BL-04 or NCFM.Undigested, unheated controls were also tested, replacing mediaautoclaved with VF with cold filtered VF. For NCFM, VF digested withα-galactosidase had the highest activity, producing 1.413% lactic acid(FIG. 5B). For BL-04, VF digested with β-glucanase had the highestactivity, producing 1.473% lactic acid (FIG. 6B). The enzyme used todigest VitaFiber™ (VF) appears to stimulate specific bacteria. VFdigested with β-glucanase stimulates BL-04, but it does not have thesame effect on NCFM. VF digested with α-galactosidase stimulates NCFM,but does not have the same effect on BL-04.

Example #4

Another prebiotic fiber source, PHGG (partially hydrolyzed guar gum),was digested with 0.2% (wt/vol) β-glucanase or 0.2% (wt/vol)α-galactosidase but showed little stimulation of either Bidfidobacteriumlactis (FIGS. 7A; 7B) or Lactobacillus acidophilus (FIG. 8A; 8B).

Example #5

Lactobacillus plantarum (LP-115), was assayed in both undigested VF, VFdigested with 0.2% (wt/vol) pectinase or a blend of 0.1% (wt/vol)pectinase plus 0.1% (wt/vol) α-galactosidase (FIGS. 9A; 9B). Theactivity of VF digested with 0.1% pectinase plus 0.1% α-galatosidase hadan activity of 1.656% lactic acid. The blended enzyme digestion had ahigher activity than pectinase alone.

Example #6

VitaFiber™ (VF) was digested with equal amounts (by weight) of twoenzymes for each strain. Bifidobacterium lactis (BL-04) was inoculatedinto VF digested with 50:50 (wt:wt), cellulase/β-glucanase (0.2% w/vol).Lactobacillus acidophilus (NCFM) was inoculated into VF digested with50:50 (wt:wt), pectinase/α-galatosidase (0.2% w/vol). LactoStim™ (0.1%)was added with the other ingredients for MRS broth (but not glucose orpolysorbate 80) following digestion. Ex. 3 (FIGS. 5A; 5B; 6A; 6B)demonstrates that β-glucanase is the preferred enzyme for growing BL-04and that α-galactosidase is the preferred enzyme for growing NCFM. VFdigested with α-galactosidase, followed by adding LactoStim™, had anactivity of 1.416% lactic acid for NCFM (FIG. 10B). The addition ofLactoStim™ thus generated a slight increase in activity, when theseresults are compared to the FIG. 5B results. VF digested withβ-glucanase followed by adding LactoStim™, had an activity of 1.365%lactic acid (FIG. 11B) for BL-04. The addition of LactoStim™ thusgenerated a slight decrease in activity, as seen when these results arecompared to the FIG. 6B results. LactoStim™ is a patented probioticstimulant protected by U.S. Pat. Nos. 8,105,576 and 8,105,577.

Example #7

Inulin was used as the fiber source in testing the growth and activityof Bifidobacterium lactic (BL-04). Inulin was digested with 0.2%(wt/vol) β-glucanase (FIGS. 12A; 12B). Lactobacillus acidophilus (NCFM)was tested with inulin or inulin digested with 0.2% (wt/vol)α-galatosidase, both with and without addition of 0.1% (wt/vol)LactoStim™ following digestion (FIGS. 13A; 13B). Digested inulin showeda small increase in activity.

Example #8

VitaFiber™ was digested with varied amounts of either a combination of50:50 (wt:wt) β-glucanase plus α-galactosidase, β-glucanase alone, orα-galatosidase alone. VF digested with half the amount of β-glucanase(0.1% w/vol) had an activity of 1.290% lactic acid for BL-04. Thisactivity was less than when 0.2% (w/vol) β-glucanase was used to digestVF (FIG. 14B). Digesting VF with 0.05% (wt/vol) β-glucanase plus 0.05%(wt/vol) α-galactosidase had an activity of 1.362% lactic acid for NCFM,while digesting VF with 0.1% (wt/vol) β-glucanase plus 0.1% (wt/vol)α-galactosidase had an activity of 1.314% lactic acid for NCFM.Digesting VF with 0.1% (w/vol) α-galactosidase alone had an activity of1.290% lactic acid for NCFM (FIG. 15B).

Example #9

VitaFiber™ was digested with β-glucanase plus α-galactosidase at either3:1 or 1:3 (wt:wt) ratios, both with and without subsequent addition ofLactoStim™. Each flask was inoculated with 0.14 gram of a 50:50 mix(wt:wt) of BL-04 plus NCFM. The highest activity occurred when VF wasdigested with 1:3 (wt:wt) β-glucanase/α-galactosidase at 0.2% (wt/vol)followed by adding 0.1% (wt/vol) LactoStim™. This resulted in anactivity of 1.512% lactic acid (FIG. 16B). This activity is higher thaneither of the highest digested VF tests assayed with a single bacterialstrain of Bifidobacterium lactis or Lactobacillus acidophilus.

Example #10

The 1:3 ratio of β-glucanase/α-galactosidase (wt:wt) at 0.2% (wt/vol)was also tested with strains of Lactobacillus salivarius (LS-33),Lactobacillus paracasei (LPC-37), Lactobacillus plantarum (LP-115),Lactobacillus rhamnosus (Lr-32), and Bifidobacterium lactis (Bi-07strain). As noted with previous experiments, Lactobacillus strains had ahigher activity when VitaFiber™ was digested with α-galactosidase ratherthan β-glucanase, and Bifidobacterium strains had a higher activity whenVF was digested with β-glucanase rather than α-galactosidase. For LS-33,LPC-37, LP-115 and Lr-32, digestion with 0.2% (wt/vol) α-galactosidasehad a slightly higher activity than with the 1:3 (wt:wt)β-glucanase/α-galactosidase blend at 0.2% (wt/vol). (FIGS. 17B, 18B,19B, 20B and 21B).

Example #11

Growth and activity of Lactobacillus rhamnosus (Lr-32), Lactobacillussalivarius (LS-33) and Lactobacillus acidophilus (NCFM) was tested withwheat dextrin or wheat dextrin digested with α-galactosidase at 0.2%(wt./vol.). (FIGS. 22B; 23B; 24B). In all cases, the digested wheatdextrin generated a higher activity (% lactic acid).

Example #12

Growth and activity of Bifidobacterium lactis (BL-04), Bifidobacteriumlactis (Bi-07) and Bifidobacterium breve (BB-03) was tested with wheatdextrin or wheat dextrin digested with β-glucanase at 0.2%(wt/vol). Inall cases, the enzyme digested wheat dextrin generated a higher activity(FIGS. 25B, 26B; 27B).

Example #13

Growth and activity of Lactobacillus plantarum (LP-115) was tested withwheat dextrin or wheat dextrin digested with either 0.2% (wt/vol)α-galactosidase or 0.2% (wt/vol) pectinase. Wheat dextrin digested with0.2% (wt/vol) α-galactosidase generated a higher activity (FIG. 28B).

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “including”, containing”, etc. are to beread expansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. It is also noted thatas used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference, and the plural include singularforms, unless the context clearly dictates otherwise. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intent in the use of such terms andexpressions to exclude any equivalent of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention as claimed.Thus, it will be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

What is claimed is:
 1. A formulation for enhancing lactobacillus growthor activity in vivo comprising α-galactosidase andisomalto-oligosaccharide.
 2. The formulation of claim 1 wherein thelactobacillus species is Lactobacillus acidophilus, Lactobacillussalivarius, Lactobacillus paracasei, Lactobacillus plantarum orLactobacillus rhamnosus.
 3. The formulation of claim 1 further includingsunflower lecithin or oleic acid.
 4. The formulation of claim 1 furtherincluding polysorbate
 80. 5. The formulation of claim 1 furtherincluding pectinase.
 6. The formulation of claim 1 further includingβ-glucanase.
 7. The formulation of claim 1 further including milkproducts including yogurt.
 8. A formulation for enhancingbifidobacterium growth or activity in vivo comprising β-glucanase andisomalto-oligosaccharide.
 9. The formulation of claim 8 wherein thebifidobacterium species is Bifidobacterium lactis (strain BL-04 orBi-07) or Bifidobacterium breve.
 10. The formulation of claim 8 furtherincluding sunflower lecithin or oleic acid.
 11. The formulation of claim8 further including polysorbate
 80. 12. The formulation of claim 8further including α-galactosidase.
 13. The formulation of claim 8further including pectinase.
 14. The formulation of claim 8 furtherincluding milk products including yogurt.
 15. A formulation forenhancing lactobacillus and bifidobacterium growth or activity in vivocomprising α-galactosidase, β-glucanase and isomalto-oligosaccharide.16. The formulation of claim 15 wherein lactobacillus species isLactobacillus acidophilus, Lactobacillus salivarius, Lactobacillusparacasei, Lactobacillus plantarum or Lactobacillus rhamnosus and thebifidobacterium species is Bifidobacterium lactis (strain BL-04 orBi-07) or Bifidobacterium breve.
 17. The formulation of claim 15 furtherincluding sunflower lecithin or oleic acid.
 18. The formulation of claim15 further including polysorbate
 80. 19. The formulation of claim 15further including pectinase.
 20. The formulation of claim 8 furtherincluding milk products including yogurt.